Synthesis of para-benzoquinone and 1,3-bis(2-chloroethyl)nitrosourea adducts and their incorporation into oligonucleotides.

Benzene is a widely known carcinogen and a cause of bone-marrow toxicity and leukaemia in humans. para-Benzoquinone is a stable metabolite of benzene. Its reaction with deoxycytidine, deoxyadenosine and deoxyguanosine produces the major stable exocyclic compounds (3-hydroxy)-1,N4-benzetheno-2'-deoxycytidine, (9-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine and (7-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine, respectively, on a large scale and at high yield. The desired products were identified by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance and UV spectroscopy. These adducts were converted to the fully protected phosphoramidites and incorporated site-specifically into a series of oligonucleotides. 1,N6-Ethano-2'-deoxyadenosine is one of the exocyclic adducts formed during DNA reaction with the antitumour agent, 1,3-bis(2-chloroethyl)nitrosourea. This compound was synthesized on a large scale with a high yield (62%) and then was converted to the phosphoramidite and incorporated site-specifically into oligonucleotides. The coupling efficiency of the incorporation of all these adducts was high (> or = 93%). After de-protection and purification of these oligomers, enzymatic hydrolysis and analysis by high-performance liquid chromatography confirmed the presence of the adduct in the oligomers. These oligomers are being used to investigate the biochemical and physical properties of these adducts.

Antisense sequence-directed cross-linking of RNA oligonucleotides by mitomycin.

Conjugates of mitomycin C (MC) and 15-mer oligodeoxyribonucleotides (ODNs) were synthesized in which the 7-amino group of MC was tethered by either a (-CH2-)6 or a (-CH2-)12 linker to the 5'-terminal phosphate of the ODNs. The conjugates were shown to be cross-linked selectively to complementary 18-mer oligoribonucleotides (ORNs). The cross-linking was dependent on reductive activation of the MC moiety of the conjugates by NADPH-cytochrome c reductase/NADPH. The cross-linked ODN-ORN hybrid duplexes were characterized as such by degeneration by RNase H. Cross-linking efficiencies of the conjugates were 50 and 25% in the case of the (-CH2-)12 tether and the (-CH2-)6 tether respectively The results demonstrate the feasibility of sequence-targeted alkylation of RNA by MC via antisense recognition.

Antisense sequence-directed cross-linking of DNA oligonucleotides by mitomycin C.

Oligodeoxyribonucleotides (ODNs) conjugated with mitomycin C (MC) via (-CH2-)n tethers of different lengths (n = 6, 12) to their terminal 5'-phosphate were synthesized, and their interaction with target complementary single-stranded DNA oligonucleotides was investigated. MC, a clinically used natural anticancer drug, is known to act as a bioreductive alkylating agent of duplex DNA with a remarkable preference for 5'-d(CG) sequences. The usual enzymatic bioreductive techniques known to trigger MC to alkylate DNA were employed in the reaction between the MC-oligonucleotide conjugates and their targets radiolabeled by 32P at their 5'-phosphate. A slow-moving radiolabeled product, detected by polyacrylamide gel electrophoresis using phosphorimaging techniques, was obtained in 15-25% yield with complementary DNA as target. Formation of these products was dependent upon complementary duplex formation. Evidence is presented that the DNA target is alkylated by the mitomycin C moiety of the ODN conjugate at the 2-amino group of a guanine base. These findings suggest that the MC-ODN conjugates may be useful specific inhibitors of cellular or viral gene expression. To our knowledge this is the first report on ODN conjugates of a reductively activated drug of known therapeutic value.

Design and synthesis of novel inhibitors of HIV-1 reverse transcriptase.

A variety of N1-substituted pyrimido[5,4-f]benzo[1,4]thiazepines, 5, designed as conformationally constrained analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymidine HEPT (1), were synthesized and evaluated for their inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The preparation of these compounds was carried out based on a Mannich-type cyclization of 6-[(2-aminophenyl)thio]uracils followed by alkylation at N1 by a one-pot Vorbruggen reaction. The pyrimidobenzothiazepines were developed to give molecules with IC50 values in the micromolar range, as exemplified by [[(2-ethoxyethyl)oxy]methyl]-pyrimido[5,4- f]benzo[1,4]thiazepine, 25, (IC50 = 0.64 microM), the most active compound of this series. The structural and electronic features of this novel class of HIV-1 RT inhibitors are presented and compared with those of HEPT (1), TIBO (2), and nevirapine (3).